What is the function of a pH buffer?

In vitro, the pH is generally maintained between 7.2 and 7.4.  All Vitrolife media are formulated to meet this narrow pH specification range, supporting optimal metabolic conditions. Maintaining a defined and physiological pH is through the inclusion of certain chemical components in the media called “pH buffers”. These pH buffers act as a weak acid or base. As a result, solutions containing such buffers can resist a change in pH caused by environmental changes.

The most commonly used pH buffer in IVF culture media is bicarbonate, which is the same buffer that is present in our blood. Carbon dioxide (CO2) in the atmosphere surrounding the culture dish will dissolve and equilibrate in the medium. Dissolved CO2 increases the amount of carbonic acid in the medium, releasing protons and therefore decreasing pH in the medium (see equation below). If the CO2 level in an incubator remains constant, the pH of the medium can be maintained.  Vitrolife culture media require a CO2 concentration of 6.0% at sea level, but the CO2 concentration can be adjusted by the end user to reach the desired pH. This ensures there is enough CO2 inside the incubator surrounding the culture dish, and in turn the formation of carbonic acid and protons, to maintain the specified pH of the culture media.

CO2 + H2O ↔ H2CO3 ↔ HCO3- + H+

When a procedure is performed in atmospheric conditions outside of the incubator, the media containing gametes and embryos are exposed to a much lower CO2 concentration (about 0.02%).  This dramatic drop in CO2 concentration will cause a change in the carbonic acid and proton content in the medium and an increase in the pH to a level above what is optimal for gametes and embryo development.  Using an oil overlay on the dish will somewhat delay the effect of this drop in CO2 concentration and subsequent pH increase.  Thus, it is recommended to use media containing a pH buffer other than bicarbonate when performing procedures under atmospheric conditions.

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G-MOPS/G-MOPS PLUS/Gx-MOPS PLUS: Medium for handling and manipulating oocytes and embryos in ambient atmosphere

G-MOPS™/G-MOPS™ PLUS/Gx-MOPS™ PLUS are MOPS (3-(N-morpholino) propane sulfonic acid) buffered media for handling and manipulating oocytes and embryos in ambient atmosphere that only needs to be heated to 37°C before use. G-MOPS/G-MOPS PLUS/Gx-MOPS PLUS must NEVER be exposed to CO2 above atmospheric level as the pH will go down below the specified range, which will harm gametes and embryos. The optimal way to prepare G-MOPS/G-MOPS PLUS/GX-MOPS PLUS is to prepare a dish with medium, covered by a layer of oil and warm the dish in an incubator or warming oven without CO2 until the medium reaches 37°C. The pH of G-MOPS/G-MOPS PLUS/Gx-MOPS PLUS is stable for several hours under oil if the temperature remains constant.

G-GAMETE: Medium for handling and manipulating oocytes and embryos in ambient atmosphere

G-GAMETE™ contains a mixture of MOPS and bicarbonate buffers and must be placed in a 6.0% CO2 incubator for equilibration.  Due to the higher bicarbonate concentration compared to what is present in G-MOPS/G-MOPS PLUS/Gx-MOPS PLUS, the pH of G-GAMETE will only be stable for approximately 7-8 minutes under atmospheric conditions. The benefit is that it can be equilibrated in a CO2 incubator.  As soon as it is exposed to atmospheric conditions the amount of dissolved CO2 in the medium will decrease and the pH will rise.

ASP: For oocyte retrieval and rinsing (follicle-flushing)
ASP™ contains a mixture of HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) and bicarbonate buffers. Similarly, to G-GAMETE, ASP needs to be equilibrated in a CO2 incubator for equilibration. What makes ASP different, is that it contains Heparin (to prevent blood clotting) and is thus ideal in circumstances where the tubes of follicular fluid may be contaminated with blood.
As far as pH is concerned, all these buffers provide a stable environment for gametes and embryos for a specified time.  However, MOPS and HEPES are nonphysiological compounds and not optimal for long time-exposure. Therefore, one should still work fast and efficiently before moving them back into the fertilization or culture medium.  It is very important to include several wash steps in the fertilization or culture medium to make sure that all the atmospheric buffer elements (i.e., MOPS or HEPES) surrounding the gametes and embryos are rinsed away and removed before they are placed back in the CO2 incubator. Failure to do so will result in fertilization and culture media having the incorrect pH.